Determinants of DOPAL Buildup in PD: Two processes efficiently prevent DOPAL from building up in dopaminergic neuronsvesicular sequestration of cytosolic DA via the type 2 vesicular monoamine transporter (VMAT2) and metabolism of DOPAL by aldehyde dehydrogenase (ALDH) to form dihydroxyphenylacetic acid (DOPAC) and to a lesser extent by aldose/aldehyde reductase to form dihydroxyphenylethanol (DOPET). The catecholaldehyde hypothesis predicts that interference with these processes increases cytosolic DOPAL levels and thereby exerts cytotoxic effects in DA-containing cells. Exploiting our laboratorys sensitive and specific methodology to measure tissue DOPAL simultaneously with other catechols, we measured endogenous DOPAL, DA, DOPAC, DOPET, and the DA precursor DOPA in a variety of brain regions in control mice, in mice with genetic manipulations of VMAT2 or ALDH genes, and in PD patients. We confirmed that putamen tissue from PD patients contains a buildup of DOPAL relative to DA and obtained evidence that putamen DOPAL buildup in PD reflects both decreased vesicular uptake of cytoplasmic DA and decreased DOPAL metabolism by ALDH (Goldstein, et al., 2013). Mechanisms of Myocardial Norepinephrine Depletion in PD: As noted above, we obtained evidence that a shift from vesicular sequestration to oxidative deamination of cytosolic catecholamines contributes to putamen DA depletion in PD. We hypothesized that an analogous shift takes place in sympathetic nerves in the heart. Whether PD entails myocardial norepinephrine (NE) depletion and a sequestration-deamination shift have been unknown. We measured apical myocardial tissue concentrations of NE, DA, and their neuronal metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC) from PD patients and controls. We devised, validated in VMAT2-Lo mice, and applied 5 neurochemical indices of the sequestration-deamination shiftconcentration ratios of DOPAC:DA, DA:NE, DHPG:NE, DOPAC:NE, and DHPG:DOPACand used a kinetic model to estimate the extent of the vesicular storage defect. The PD group had decreased myocardial NE content (p<0.0001). The majority of patients (70%) had severe NE depletion (mean 2% of control), and in this subgroup all 5 indices of a sequestration-deamination shift were increased compared to controls (p<0.001 for each). Vesicular storage in residual nerves was estimated to be decreased by 84-91% in this subgroup. Therefore, most PD patients have severe myocardial NE depletion, due to both sympathetic denervation and decreased vesicular storage in residual nerves (Goldstein, Sullivan, et al., 2014). . Genetic and Environmental Determinants of Abnormal Catecholamine Metabolism in PD Animal Models: In a collaborative study with J. Casida (UC Berkeley), we found that the fungicide benomyl potently and rapidly inhibits ALDH and builds up DOPAL in vivo in mouse striatum and in vitro in PC12 cells and human cultured fibroblasts and glial cells. The in vivo results resembled those we reported previously with knockouts of the genes encoding ALDH1A1 and 2, a mouse model of aging-related PD. Exposure to specific pesticides may therefore increase PD risk via ALDH inhibition and consequent DOPAL buildup (Casida, et al., 2014). This was the cover article in Chem Res Toxicol. In a collaborative study with G. Miller (Emory) on neuropathologic consequences of very low VMAT2 activity, we obtained evidence for acceleration of aging-related loss of noradrenergic neurons in the locus ceruleus, the main source of NE in the brain, as well as in the heart in VMAT2-Lo mice (Taylor, Alter, Wang, Goldstein, & Miller, 2014). Mice with elevated VMAT2 activity, on the other hand, have enhanced DA release and decreased PD-related neurodegeneration in vivo (Lohr, et al., 2014). The latter study was a featured article in PNAS. A potentially important link between catecholaldehydes and synucleinopathy in the pathogenesis of PD is DOPAL-induced oligomerization of alpha-synuclein. We found that divalent metal cations, especially Cu+2, augment this oligomerization (Jinsmaa, et al., 2014). The findings fit with the notion of neuronal damage from dysregulation of catecholamine metabolism, alpha-synucleinopathy, and divalent metal cations. Parkinsonism and Putamen DOPAL Buildup without Intra-neuronal Synuclein Deposition in Multiple System Atrophy (MSA): In PD, immunoreactive alpha-synuclein (SYN-ir) is found in Lewy bodies, whereas in MSA SYN-ir is found in glial cytoplasmic inclusions (GCIs). A minority of MSA patients have both GCIs and cardiac sympathetic denervation. It has been thought that such patients have a hybrid disease, in which pathogenic alpha-synuclein deposits are found in both glial cells and sympathetic neurons. In our patient, who clinically had the Parkinsonian form of MSA, brain PET scanning showed bilaterally decreased putamen 18F-DOPA-derived radioactivity and thoracic PET scanning showed markedly decreased 18F-dopamine-derived radioactivity throughout the left ventricular myocardium. At autopsy the patient had putamen atrophy and drastic myocardial norepinephrine and putamen dopamine (DA) depletion. SYN-ir deposits were noted in glia, but there was no SYN-ir deposition in neurons or sympathetic ganglia. Putamen DA:DOPA and DOPAC:DOPAL ratios were low and DOPAL:DA high. This important case demonstrates that in MSA catecholamine depletion can occur without Lewy bodies or intra-neuronal alpha-synuclein deposition in the sympathetic nervous system or brain. Meanwhile, the pattern of catechol ratios suggests the same triad of a sequestration-to-oxidative deamination shift, DOPAL buildup, and decreased DOPAL detoxification by aldehyde dehydrogenase as we have found in sporadic PD. Therefore, in MSA, the putative catecholamine autotoxicity pattern can occur without intra-neuronal synucleinopathy (Cook, Sullivan, Holmes, & Goldstein, 2014).